1. Field of the Invention
The present invention relates to the field of labeled activity-based probes which bind to active cysteine proteases, including cathepsins and caspases, and the use of such probes as radioactive imaging agents in vivo.
2. Related Art
Proteases play important roles in the regulation of both normal and disease processes. The cysteine protease family comprises six major families: the papain family, calpains, clostri-pains, streptococcal cysteine proteases, viral cysteine proteases and most recently established, caspases (also called apopains). In particular the papain family cysteine cathepsins are frequently over-expressed at the mRNA, protein and activity levels in a number of human cancers such as glioma, melanoma, breast, colorectal, gastric, lung, and prostate carcinomas. In addition, expression of a number of cysteine cathepsins, including cathepsin B and L, is increased in pre-neoplastic lesions and changes in both localization and subcellular distribution of these proteases are often observed in tumors. This combination of increased expression, activity and altered localization of these enzymes as well as their positive association with tumor progression, metastatic potential and disease outcome make them potentially valuable cancer biomarkers. The cathepsin family of lysosomal protease includes the cysteine proteases; cathepsins B, H, K, L, W, C, F, V, X, and S, and the aspartyl protease; cathepsins D, and the serine protease cathepsin G. The various members of this endosomal protease family are differentially expressed. Some, such as cathepsin D, have a ubiquitous tissue distribution, while others, such as cathepsin S, are found mainly in monocytes, macrophages, and other cells of the immune system. The activity of cysteine proteinases is optimal at pH values of <7, as found in lysosomes, where these enzymes perform their main biological function. However, there is increasing evidence for extracellular functions of cathepsins produced by macrophages, osteoclasts, fibroblasts, and transformed cells into specific pericellular locations and that these proteases can function well in neutral pH environments as well, (See, Oksjoki et al., “Differential expression patterns of cathepsins B, H, K, L and S in the mouse ovary,” Molecular Human Reproduction, Vol. 7, No. 1, 27-34, January 2001.)
In addition, the present imaging and labeling agents may be directed against caspases. Initiator caspases (e.g., CASP2, CASP8, CASP9 and CASP10) cleave inactive pro-forms of effector caspases, thereby activating them. Effector caspases (e.g., CASP3, CASP6, CASP7) in turn cleave other protein substrates within the cell resulting in the apoptotic process. Such probes can be useful in imaging active apoptotic processes in a cell.
Molecular imaging is a valuable new technology that is helping to provide a better understanding of cancer and other diseases. Novel imaging methods and molecularly targeted tracers can now be used to not only to locate a tumor, but also to visualize the expression and activity of specific molecular targets and biological processes in a tumor. These imaging methods have the potential to facilitate both early disease detection and to aid in the process of drug development. Directed targeting of enzymatic proteins such as protease using imaging agents has the potential to provide even more detail about the basic biological framework of a tumor and also provide better resolution of the disease phenotype. In addition since most proteases are initially synthesized as inactive zymogens that are activated by a complex set of post-translational mechanisms, tools that report on enzyme activity and not simple protein abundance will be required to fully understand their function in complex biological processes. For this reason, a number of recent studies have focused on the development of protease directed imaging agents that act as substrates for a target enzyme. This approach takes advantage of the catalytic nature of the target in order to amplify a signal from a reporter substrate. Strategies that make use of quenched fluorescent substrates and also fluorescent molecules that only penetrate a cellular membrane when processed by a target protease have been developed. While all of these methods have provided valuable new tools for the optical imaging of protease activity in vivo, none of these methods have been translated for use in radiological imaging.
Described and exemplified below is the development and application of radiolabeled small molecule activity based probes that can be used for positron emission tomography (PET) imaging of cysteine cathepsin and caspase activity. These probes have a reactive group as in the peptide acyloxymethyl ketone (AOMK) probes that have been reported to be highly selective labels of a number of classes of cysteine cathepsins. This class of activity-based probes has also recently been used for optical imaging of cysteine cathepsin activity in live cells (Blum et al. Nat Chem Bio 2005, below) and in near-infrared (NIRF) labeled form for non-invasive optical imaging of cysteine cathepsin activity in living subjects. The present, novel probes couple the intrinsic advantages of nuclear imaging including high sensitivity, capability of quantification, and clinical translation with the specific, covalent nature of these small molecule imaging agents. In addition, their relatively small size compared to substrate based probes gives them favorable in vivo pharmacodynamic properties and cellular uptake. In examples below, it is shown that a 64Cu-labeled derivative of the previously reported cysteine cathepsin probe GB111 (disclosed in U.S. patent application Ser. No. 11/502,255 filed Aug. 10, 2006) is taken up by tumors and provides good signal to background ratios. Furthermore, the relative levels of probe labeling in tumors directly correlates with the activity levels of cathepsins B and L in those tumors as demonstrated by biochemical profiling methods. Thus, these new probes represent potentially valuable imaging tools for application to a number of clinically relevant diseases that involve overexpression of cysteine cathepsin activity.
Specific Patents and Publications
Kato D, Boatright K M, Berger A B, Nazif T, Blum G, Ryan C, Chehade K A, Salvesen G S, Bogyo M, “Activity-based probes that target diverse cysteine protease families,” Nat Chem Biol, 2005; 1: 33-8, describes a series of quenched near-infrared fluorescent activity-based probes (qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown previously to be important in multiple stages of tumorigenesis. These ‘smart’ probes emit a fluorescent signal only after covalently modifying a specific protease target. This paper discloses the compound Z-FR-AOMK shown in FIG. 1 of the present application. The Z-FR-AOMK was active only against cathepsin B, not cathepsins Z, H, J, H or C. It also gives a detailed synthetic method, which may be adapted according to the teachings below to make compounds of the present invention.
Weissleder et al., “In vivo imaging of tumors with protease-activated near-infrared fluorescent probes,” Nature Biotechnology, 17, 375-378 (1999) describe the imaging of tumor-associated lysosomal protease activity using autoquenched near-infrared fluorescence (NIRF) probes. The authors used a synthetic graft copolymer consisting of poly-L-lysine sterically protected by multiple methoxypolyethylene glycol (MPEG) side chains.
Blum et al., “Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes,” Nature Chemical Biology, 3, 668-677 (2007) disclose a series of quenched near-infrared fluorescent activity-based probes (qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown previously to be important in multiple stages of tumorigenesis. These ‘smart’ probes emit a fluorescent signal only after covalently modifying a specific protease target. As a starting point, the authors (which include present inventors) used the peptide acyloxymethylketones (AOMKs) GB111 and GB117 that were recently described as tools for cell-based imaging of cysteine cathepsin activity (see paper cited below).
Blum, G. et al. Dynamic imaging of protease activity with fluorescently quenched activity-based probes. Nat. Chem. Biol., 1, 203-209 (2005), discloses the acyloxymethyl ketone (AOMK) reactive group for probe design, as this ‘warhead’ targets diverse families of cysteine proteases. More importantly, the mechanism of covalent modification of a cysteine probes based on the acyloxymethyl ketone (AOMK) reactive group for probe design, which targets diverse families of cysteine proteases. Those probes target the papain family of cysteine proteases, as this family has been extensively studied with ABPs and a number of cell-permeable reagents have successfully been designed. The authors (which include present co-inventors) carried out the synthesis of the resulting quenched probe GB117 and its corresponding nonquenched control GB111 using a combination of solid and solution-phase chemistries.
U.S. Pat. No. 6,475,485 discloses two novel human cathepsin proteins, referred to as HCP-1 and HCP-2 and, collectively, HCPs, which share features with other proteins involved in proteolysis.
Joyce et al., “Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis,” Cancer Cell, Vol 5, 443-453, May 2004, disclose a broad-spectrum cysteine cathepsin inhibitor which was used to pharmacologically knock out cathepsin function at different stages of tumorigenesis, impairing angiogenic switching in progenitor lesions, as well as tumor growth, vascularity, and invasiveness.